Impact of cryopreservation on CAR T production and clinical response.

Adoptive cell therapy with chimeric antigen receptor (CAR) T cells has become an efficient treatment option for patients with hematological malignancies. FDA approved CAR T products are manufactured in centralized facilities from fresh or frozen leukapheresis and the cryopreserved CAR T infusion product is shipped back to the patient. An increasing number of clinical centers produce CAR T cells on-site, which enables the use of fresh and cryopreserved PBMCs and CAR T cells. Here we determined the effect of cryopreservation on PBMCs and CD19 CAR T cells in a cohort of 118 patients treated with fresh CAR T cells and in several patients head-to-head. Cryopreserved PBMCs, obtained from leukapheresis products, contained less erythrocytes and T cells, but were sufficient to produce CAR T cells for therapy. There was no correlation between the recovery of PBMCs and the transduction efficacy, the number of CAR T cells obtained by the end of the manufacturing process, the in vitro reactivity, or the response rate to CAR T therapy. We could show that CAR T cells cryopreserved during the manufacturing process, stored and resumed expansion at a later time point, yielded sufficient cell numbers for treatment and led to complete remissions. Phenotype analysis including T cell subtypes, chemokine receptor and co-inhibitory/stimulatory molecules, revealed that fresh CAR T cells expressed significantly more TIM-3 and contained less effector T cells in comparison to their frozen counterparts. In addition, fresh CAR T infusion products demonstrated increased in vitro anti-tumor reactivity, however cryopreserved CAR T cells still showed high anti-tumor potency and specificity. The recovery of cryopreserved CAR T cells was similar in responding and non-responding patients. Although fresh CAR T infusion products exhibit higher anti-tumor reactivity, the use of frozen PBMCs as staring material and frozen CAR T infusion products seems a viable option, as frozen products still exhibit high in vitro potency and cryopreservation did not seem to affect the clinical outcome.

Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.

The gut microbiome has been shown to influence the response of tumors to anti-PD-1 (programmed cell death-1) immunotherapy in preclinical mouse models and observational patient cohorts. However, modulation of gut microbiota in cancer patients has not been investigated in clinical trials. In this study, we performed a phase 1 clinical trial to assess the safety and feasibility of fecal microbiota transplantation (FMT) and reinduction of anti-PD-1 immunotherapy in 10 patients with anti-PD-1-refractory metastatic melanoma. We observed clinical responses in three patients, including two partial responses and one complete response. Notably, treatment with FMT was associated with favorable changes in immune cell infiltrates and gene expression profiles in both the gut lamina propria and the tumor microenvironment. These early findings have implications for modulating the gut microbiota in cancer treatment.